Monoclonal antibody specific for ppar gamma, hybridoma cell line producing the same, and method for detecting regulator related to diseases, including inflammation, cancer and metabolic diseases, using the same

ABSTRACT

The present invention relates to a hybridoma cell line producing a PPAR gamma-specific monoclonal antibody, and a method for detecting a PPAR gamma ligand related to the progression of difficult diseases, such as cancer, inflammation and metabolic diseases obesity and diabetes), using the PPAR gamma-specific monoclonal antibody. This PPAR gamma-specific monoclonal antibody and the method for screening a PPAR gamma ligand using the monoclonal antibody will be commercially used for screening a PPAR gamma regulator related to diseases such as inflammatory, cancer and metabolic diseases, and also will serve as an useful tool for analyzing the function of such a ligand.

TECHNICAL FIELD

The present invention relates to a PPAR gamma-specific monoclonalantibody, a hybridoma cell line producing the same, and a method fordetecting a regulator related to diseases including inflammation, cancerand metabolic diseases, using the same.

BACKGROUND ART

The protein PPAR (peroxisome proliferator activated receptor) is knownas a nuclear receptor. Peroxisome which is an intracellular organelle isinvolved in oxidation and present in the liver and kidneys at largeamounts. Also it oxidizes an intracellular fatty acid to produceperoxides which serve to neutralize toxic substances. In addition, ithas a function of decomposing excess hydrogen peroxide into water andoxygen by catalase, an oxidase. A peroxisome proliferator signifies acompound capable of increasing the number of peroxisomes, and includesfat and fatty acid, and fibrate and prostaglandin ashyperlipemia-treating agents.

Nuclear receptors having such peroxisome proliferators as ligands arecollectively called PPARs, which are divided into PPAR alpha, PPARdelta/beta and PPAR gamma. As known in the art, PPAR alpha is mainlyexpressed in the liver, and involved in the oxidation of fatty acids orthe neutralization of toxic substances, and related to inflammatoryreaction. PPAR delta/beta is found to distribute uniformly and to beinvolved in embryonic development, and PPAR gamma is known to beinvolved in the differentiation and accumulation of fat cells.

It was recently found that the expression of PPAR gamma was increased ina differentiation process of a fat cell line into a fat cell, and thatexpression of PPAR gamma in fibroblast having no differentiation abilityresulted in differentiation of the fibroblast into a fat cell.Particularly, PPAR gamma2 is divided into two isoforms and reported tobe specifically expressed only in fat cells at large amounts. mRNA ofmouse PPAR gamma1 is coded by eight exons, whereas mRNA of PPAR gamma2is coded by seven exons. The 5′ untranslated sequence of mRNA of mousePPAR gamma1 is coded by two exons, whereas the 5′ untranslated sequenceof PPAR gamma2 and the additional N-terminal amino acids are coded byone exon. The two isoforms are yielded by alternative promoter use anddifferent splicing, and increase the variety of ligands and maketissue-specific expression possible (Zhu Y et al., 1995).

Meanwhile, type 2 diabetes is characterized by the insulin resistance ofskeletal muscle tissues, liver tissues and fat tissues, etc. In theearly 1980's, among treating agents of type 2 diabetes, glitazones andthe like belonging to thiazolidinediones (TZD) were first reported as adrug which allows glucose level to be reduced and insulin resistance tobe improved without stimulating insulin secretion in an experimental ratmodel of type 2 diabetes.

As glitazones, treating agents of type 2 diabetes, are found to be PPRAgamma agonists (Lehmann J M et al, 1995), it is reported that PPRA gammaligands (agonists) can improve insulin resistance.

This fact seems to prove the therapeutic effect of the PPAR gammaagonists and to suggest the utility of a system for screeningdiabetes-effective substances by ELISA, which was established by thepresent inventors.

Meanwhile, in order to examine how the activity of PPAR gamma in fatcells influences the metabolism of glucose in the muscles and liver, themechanisms of PPAR gamma agonists as agents for improving diabetes willnow be described. First, PPAR gamma protein in fat cells is known asregulating the release of endocrine signal molecules influencing themetabolism of glucose in the muscles and liver, such as cytokine TNF-αor leptin. Expression of the two signal molecules is inhibited by thePPAR gamma agonists in fat cells, and it was found that TNF-α resultedin the increase of insulin resistance and leptin interfered-with insulinsignal transmission in any cells (Cohen B. et al., 1996; and Muller G.et al., 1997). This seems to increase insulin resistance. Thus, insulinresistance caused by such two signal molecules can be improved by thePPAR gamma agonists.

Second, there is the antihyperglycemic effect of PPAR gamma agonists.Generally, glucose and fatty acids compete with each other for an energysubstrate in muscles, so that the increase of the amount of fatty acidsresults in the decrease of glucose consumption. Thus, it is believedthat the increase of free fatty acids and the increase of glucosesynthesis or gluconeogenesis are connected with each other. In thiscase, the PPAR gamma agonists stimulate fat cells to absorb and storefatty acids, thereby reducing the amount of circulating triglyceridesand free fatty acids. Furthermore, the PPAR gamma agonists are known ashaving an indirect effect on glucose metabolism, reducing the level offatty acids in the muscles or liver (Martin G. et al., 1998).Accordingly, the PPAR gamma agonists stimulate fatty acid flow from themuscles or liver to white fatty acids and show a dramatic effect onenergy expenditure, thereby causing the reduction of gluconeogenesis inthe liver and the increase of glucose consumption in muscles.

Finally, PPAR gamma is also expressed in the muscles and liver at alower expression level than in fat cells and can show an effect ofimproving diabetes by its direct activation. Namely, it is reported thattreatment of experimental rats deficient in fat tissues withtroglitazone as a PPAR gamma agonist reduces hyperglycemia and increasesinsulin sensitivity. This mechanism can be regarded as the role of PPARgamma agonists generated by a pathway separate from fat cells (Burant CF et al., 1997).

And the PPAR gamma agonists show a direct or indirect effect in varioustissues including the muscles and liver.

Moreover, for atheromatous lesions, PPAR gamma is known as beingexpressed in macrophages including foam cells at a high level.

The foam cells generally means cholesterol-laden cells converted frommacrophages embedded in the inner arterial wall. This conversion of themacrophages into the foam cells is regarded as a definite symptom ofoccurrence of arteriosclerosis.

The conversion process of the cells is known as having a connection withthe internalization of oxLDL particles by scavenger receptors, such asCD36 (cell adhesion molecule) and scavenger receptor-A. It was recentlyreported that PPAR gamma was also closely connected with this process(Tontonoz P. et al., 1998).

For example, it is known that treatment of a human acute monocyticleukemia cell line (THP-1) with PPAR gamma and RXR alpha (retinoid Xreceptor alpha) agonists induces the expression of PPAR gamma2 and CD36and promotes the absorption of oxLDL, and treatment of the aorta ofexperimental rats with the PPAR gamma agonist increases the expressionof CD36. Particularly, 9-HODE and 13-HODE, two components of oxLDL, werefound to be PPAR gamma agonists. Accordingly, PPAR gamma is an importantcomponent of oxLDL-PPAR gamma-CD36 contributing to the accumulation oflipids induced from oxLDL by macrophages. These results indicate thatPPAR gamma agonists have the possibility of promoting the formation offoam cells, but clinical data show that glitazone protects patients withtype 2 diabetes from arteriosclerosis which can frequently secondarilyoccur in these patients. This is because treatment of low densitylipoprotein (LDL) receptor-deficient rats (arteriosclerosis model) withrosiglitazone and GW784 is known as inhibiting the formation ofatheromatous lesion in spite of the increase of CD36 expression.

According to recent studies, it was found that the release ofcholesterol from macrophages was controlled by ABCA1 (ATP bindingcassette A1), a member of ATP binging cassettes (ABC) ofenergy-dependent transporter proteins, in which ABCA1 is mutated inpatients with tangier disease caused by cholesterol accumulation inmacrophages and other reticuloendothelial cells. The transcription of anABCA1 gene is regulated by a nuclear oxysterol receptor LXR (liver Xreceptor), and PPAR gamma and LXR agonists act together to induce theexpression of ABCA1 and to promote the release of cholesterol frommacrophages and THP-1 cell lines induced from rat embryonic stem cells(Chawla A. et al., 2001).

Furthermore, the ABCA1 gene is known as a direct target gene of a PPARgamma/RXR heterodimer and causes the expression of LXR-alpha when theactivity of PPAR gamma is increased. This leads to the increase of ABCA1expression and the release of cholesterol. In addition, PPAR gamma isknown to regulate the introduction and release of cholesterol esters inmacrophages. By this effect, oxLDL will be removed which increases therelease of free cholesterol introduced to the liver, and causes aorticlesions by absorption into macrophages. Particularly, the PPAR gammaagonists seem to interfere with the development arteriosclerosis in vivoby producing HDL (high density lipoprotein) in the human and increasingthe release of cholesterol from macrophages and endothelial cells.

Moreover, there is the broad role of PPAR gamma in the regulation ofinflammatory reaction of monocytes/macrophages.

Treatment of PPAR gamma including 15d-PGJ2 and glitazone, with monocytesor macrophages, reduces the expression of pro-inflammatory cytokine suchas TNF-alpha and IL-6 and inhibits the activity of macrophages. However,the PPAR gamma agonists induce an effect of inhibiting inflammatoryreaction at a different concentration from a concentration required toactivate PPAR gamma in cell-based assays. In addition, any potentialPPAR gamma agonists have no effects, but 15d-PGJ2 is the most powerfulinhibitor against the cytokine production of monocytes or macrophages invitro. This suggests that the anti-inflammatory effect of the PPAR gammaagonists can be mediated by PPAR-independent mechanism. This hypothesisis supported by several recent studies. Namely, it was found thatglitazone inhibited cytokine production in rats treated with LPS(lipopolysaccharide) and that several different PPAR gamma agonists(15D-PGJ2 and rosiglitazone) inhibited cytokine production even inembryonic stem cells having PPAR gamma+/+ or PPAR gamma± or PPARgamma−/−, as in macrophages.

Meanwhile, 15d-PGJ2 was recently found to inhibit NF-κB activity (RossiA. et al., 2000; and Straus D. S. et al., 2000). NF-κB causes acuteinflammatory reaction by the covalent modification of its DNA-bindingdomain and IκB kinase as its regulatory subunit. This suggests that PPARgamma is not substantially effective against acute inflammations causedby leucocytes.

Furthermore, hypertension is one of metabolic defects which oftenaccompany obesity and type 2 diabetes, etc. Its pathogenesis is complexand connected with blood pressure dysregulation, insulin sensitivity,vascular function, and lipid metabolism.

Recent genetic analysis shows that a PPAR gamma dominant negative mutantis connected with severe hypertension Treatment of an animal model ofhypertension with PPAR gamma agonists shows low blood pressure, but itis not yet known that the PPAR gamma agonists are involved in amechanism forming the basis of an anti-hypertensive effect. However, thefact glitazone reduces blood pressure in the human with no diabetes andan animal model of hypertension having no connection with insulinresistance indicates that the anti-hypertensive effect of PPAR gammaagonists is independent of insulin-sensitizing actions.

Since PPAR gamma is expressed in intra-vascular endothelial cells, thePPAR gamma agonists are considered as improving hypertension byregulating the expression of vascular factors connected with themaintenance of vascular tone, such as type C nutriuretic peptide,endothelin, and plasminogen activator inhibitor-1 (Itoh H. et al.,1999).

Furthermore, the proliferation inhibition and pro-differentiationeffects of the PPAR gamma agonists suggest that these compounds can beused as an agent for inhibiting the proliferation of de-differentiatedtumor cells. This hypothesis supports the experimental results showingthat transplantation of BNX triple immuno-deficient nude mice withbreast cancer cells (Elstner E. et al., 1998) and prostate tumor cells(Kubota T. et al., 1998) followed by treatment with TZDs inhibits theproliferation of such tumor cells.

These effects show that a differentiation program according to theresult of PPAR-mediated activation can be used even in nonadipogeniclineage colonic cells and inhibits the development of cancer Incontrast, when a transformed experimental mouse deficient in one copy ofa gene coding for an APC (adenomatous polyposis coli tumor suppressor istreated with PPAR gamma agonists of a significantly higher amount thanone required to increase insulin activity, the PPAR gamma agonistspromote the development of colonic tumor (Lefebvre A. M. et al. 1998). Ahuman colon cancer cell line studied by Sarraf and his research team hasboth normal and malfunctioning APCs, and it is considered that manyunknown factors required for colonic cell proliferation in anexperimental rat model are involved therein.

Furthermore, a recent study conducted by Pilot showed that administeringthe PPAR gamma agonists to patients with solid liposarcoma causedantineoplastic pro-differentiation. These agonists reduce theproliferation rate of cancer cells, and thus, it is expected that theywill make the progression of this disease slow. In any human coloncancers, it was observed that functions of PPAR gamma were lost due tothe PPAR gamma mutation (Sarrf P. et al., 1999). The PPAR gamma agonistsinduce growth arrest and also the synthesis of different markers ofhuman colon cancer cells in cell culture. This discovery suggests thatPPAR gamma inhibits cellular transformation.

Moreover, it is found that the PPAR agonists can be used as inhibitorsagainst angiogenesis, a process necessary for solid-tumor growth, and ametastasis process. Thus, such evidences show that PPAR gamma activationinhibits the growth and development of cancer, and PPAR gamma ligands oragonists will provide a new aim for therapeutic application.

Meanwhile, immunoassays are used for detecting such substances.

Generally, immunoassays using antigen-antibody reaction are known asmethods of qualitatively and quantitatively analyzing a biologicalsubstance to be measured, in which a specific antibody to an antigensubstance to be measured is made such that it can be bound to theantigen, and the binding of the antibody to the antigen is measured withvarious labels which can recognize and measure the antigen-antibodycomplex with a device.

Immunoassays of biological substances which have been used till now canbe divided according to the kind of used labels into radioimmunoassay(RIA) using radioactive isotopes as labels, and non-radioactiveimmunoassays, including enzyme-linked immunosorbent assay (ELISA) usingenzymes or fluorescent substances as labels, and fluorescence enzymeimmunoassay (FEIA).

The radioimmunoassay among such immunoassays is widely used which showshigh sensitivity and is carried out in a precise, simple, easy and rapidmanner. Also it has another advantage in that instruments, such as gammaand beta counters, which can be used in this method, are not soexpensive.

However, the greatest shortcoming of the immunoassay using radioactiveisotopes is that radioactive wastes are released at large amounts. Alsothis immunoassay is disadvantageous in that the amount of use and thekind of radioactive isotopes are limited by regulations.

In order to solve such problems, enzyme-linked immunosorbent assay(ELISA) is used which is divided into direct ELISA and indirect ELISAaccording to a labeling type.

Direct ELISA is a way of conducting direct labeling to an antibody or anantibody fragment by a crosslinker, and indirect ELISA is a way ofbinding hapten to an antibody and conducting measurement using a labelrecognizing this complex.

Examples of the crosslinker which is used in the direct ELISA includesN,N′-orthophenylenedimaleimide,4-(N-maleimidomethyl)cyclohexane-N-succinimide ester,6-maleimidohexane-N-succinimide ester, 4,4-dithiopyridine and the like.Examples of hapten which is used in the indirect ELISA include biotin,dinitrophenyl pyridoxal, fluoresamine and the like, in which biotin usesavidin or streptoavidin as a recognition ligand.

Horseradish peroxidase is mainly used as an enzyme in ELISA, because itcan react with many substrates and can be easily bound to an antibody.

In order to verify a labeled enzyme, horseradish peroxidase useshydrogen peroxide (H₂O₂) as a substrate solution, and2,2′-azino-di-[3-ethylbenzothiazoline sulfonic acid] ammonium salt(ABTS), 5-aminosalicylic acid, orthophenylenediamine, 4-aminoantipyrine,or 3,3′,5,5′-tetramethylbenzidine as a color developer. Also, alkaliphosphatase employs orthonitrophenyl phosphate or paranitrophenylphosphate as a substrate, and β-D-galactosidase usesfluorescein-di-(β-D-galactopyranoside) or 4-methylumbellifery as asubstrate.

Meanwhile, if a substance immobilized to a plate is an antibody, ELISAis also called a sandwich ELISA.

DISCLOSURE OF INVENTION

Accordingly, an object of the present invention is to provide a specificmonoclonal antibody for PPAR gamma, and a hybrodoma cell line producingthe same.

Another object of the present invention is to provide a method fordetecting a regulator related to inflammation, cancer and metabolicdiseases using a specific monoclonal antibody for PPAR gamma, and a kitfor use in the method.

To achieve the above-mentioned objects, in one aspect, the presentinvention provides a Pγ48.34A cell line (KCTC 10482BP), which produces amonoclonal antibody whose immunogloblulin subtype is G_(2a), themonoclonal antibody having having specific immunoreactivity for humanand mouse PPAR gamma proteins.

In another aspect, the present invention provides a monoclonal antibodyproduced by this cell line.

In still another aspect, the present invention provides a method fordetecting a regulator related to cancer, inflammation and metabolicdiseases, the method comprising the Steps of: coating a PPAR gammacoactivator on a plate; adding a sample containing a PPAR gamma proteinand a candidates related to cancer, inflammation and metabolic diseasesto the plate; adding a monoclonal antibody to the plate so that theantibody binds to the PPAR gamma protein; adding an antibody havingimmunoreactivity for the monoclonal antibody and labeled with an enzymeto the plate; and detecting the label and measuring the concentration ofthe PPAR gamma protein, thereby determining if the regulator related tocancer, inflammation and metabolic diseases is present in the sample.

In yet another aspect, the present invention provides a kit fordetecting a ligand related to cancer, inflammation and metabolicdiseases by immunoassay, the kit comprising: a coactivator proteincoated on a plate; a PPAR gamma protein capable of binding to thecoactivator protein; a monoclonal antibody having specificimmunoreactivity for the PPAR gamma protein; and an antibody labeledwith an enzyme and having immunoreactivity for the monoclonal antibody.

Preferably, the PPAR gamma protein is PPAR gammal or PPAR gamma2 , andthe coactivator is SRC-1 (steroid receptor coactivator).

Hereinafter, the present invention will be described in detail.

The present invention provides a specific monoclonal antibody for PPARgamma related to inflammation, cancer and metabolic diseases (obesityand diabetes), and a hybridoma cell line producing the monoclonalantibody. Also the present invention provides a method for detecting aregulator capable of regulating the PPAR gamma activity, by means of thespecific monoclonal antibody for the PPAR protein, and a kit using thismethod.

According to the present invention, in order to produce the specificmonoclonal antibody for PPAR gamma and the hybridoma cell line producingthe same, PPAR gamma2 cDNA was isolated from human lipoma, amplified andpurified. After cloning into an E. coli expression vector, it isexpressed into a fusion protein (hereinafter, referred to as His-PPARgamma2 and GST-PPAR gamma2) in E. coli, and isolated and purified onnickel beads, thereby producing an antigen for the antibody.

And, a His-PPAR gamma2 soluble protein is emulsified with an equalamount of a Freund's complete adjuvant and inoculated into the abdominalcavity of a mouse required for the development of a hybridoma cell line.After two weeks, to increase immunity, the mouse receives a boosterinjection, in which a His-PPAR gamma2 soluble protein is mixed with anequal amount of a Freund's incomplete adjuvant and injected into theabdominal cavity of the mouse in two times.

A spleen cell taken from the immunized mouse and a NS-1 myeloma cell aremixed with each other at the ratio of 10:1, and fused by the addition ofpolyethylene glycol. The cells are cultured in HAT medium, and when thegrowth of the fusion cells is continuously confirmed, the cells areproliferated in HT medium. Among the cells growing in the HAT medium,cells which specifically react only with the PPAR gamma recombinantprotein are screened by enzyme-linked immunosorbent assay (ELISA).

The screened hybridoma cell line is cloned as a monoclone by a limitingdilution technique, thereby establishing a cell line consisting of cellsproliferated from one cell.

The established cell line of the present invention was termed Pγ48.34A,and deposited under the accession number KCTC 10482BP on Jun. 3, 2003with the Korean Collection for Type Cultures (KCTC).

After the cell line is injected into the abdominal cavity of the mouse,ascitic fluid is taken from the mouse, thereby producing the monoclonalantibody of the present invention at large amounts.

The isotype of the PPAR gamma protein-specific monoclonal antibody,which is secreted by the cell line established in the present invention,is an immunoglobulin G_(2a) (IgG_(2a)) isotype. As confirmed by Westernblotting and immunoprecipitation, the cell line developed by the presentinventors shows high specificity for human and mouse PPAR gammaproteins. Also, as confirmed by Western blotting of other commerciallyavailable specific monoclonal or multiclonal antibodies for PPAR gamma,the cell line of the present invention has no cross-reactivity withother isotypes of PPAR, such as PPAR alpha, delta and beta.

According to the present invention, an enzyme-linked immunosorbent assay(ELISA) method was constructed. In this ELISA, the conformation of PPARgamma in the binding of PPAR gamma and SRC-1 protein known as an agonistfor PPAR gamma is induced, and using properties of agonist or antagonistligands influencing the binding between PPAR gamma and SRC-1, aregulator related to inflammation, cancer and metabolic diseases isdetected from a mixture containing the monoclonal antibody, the PPARgamma recombinant protein, the SRC-1 recombinant protein and variouscandidates such as natural and artifactual compound.

According to the present invention, the SRC-1 recombinant protein isisolated from E. coli, purified, and attached to a microtiter plate andthen reacted with a human PPAR gamma2 fraction expressed into GST- andHis-fusion proteins in E. coli., so that a change in binding activitycaused by transformation of the PPAR gamma protein with naturalsubstances and various chemicals is examined. As a result, it was foundthat the use of the specific monoclonal antibody for PPAR gamma,developed by the present inventors, allowed a PPAR gamma regulator to bedetected in a more precise and reliable manner than other commerciallyavailable specific antibodies for PPAR gamma.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows the analysis of amplified cDNA of PPAR gamma2 on 1%agarose gel. The cDNA was obtained by isolating mRNA from human lipoma,reverse-transcripting mRNA to cDNA using reverse transcriptase andamplifying cDNA with PPAR gamma2 primers.

FIG. 1B is a schematic view showing that PPAR gamma2 is cloned into anE. coli expression vector to mass-produce the PPAR gamma2 protein in E.coli.

FIG. 1C shows the results of analysis of His-fused PPAR gamma2 on 12%SDS-PAGE. The His-fused PPAR gamma2 was amplified, isolation andpurified in E. coli.

FIG. 2A shows that a monoclonal antibody has a high affinity forGST-fused PPAR gamma2 as an antigen.

FIG. 2B shows the isotype of a PPAR gamma-specific monoclonal antibody,which was examined by an isotyping kit (Immuno-Type™ mouse monoclonalantibody isotyping kit, BD science).

FIG. 3A shows the result of Western blotting analysis of human and mouseHis-fused recombinant proteins.

FIG. 3B shows the results of Western blotting analysis indicating that aPγ48.34A antibody developed by the present inventors shows non-specificreaction with skimmed milk and BSA whereas some of existing PPARgamma-specific antibodies react with skimmed milk and BSA.

FIG. 4A shows the results of the Western blotting analysis of PPAR gammausing a mouse 3T3-L1 cell line.

FIG. 4B shows that a PPAR gamma-specific antibody (Pγ48.34A) can also beused in immunoprecipitation of a 55 kDa PPAR gamma regardless of fatdifferentiation.

FIG. 5A shows the non-specific binding of a Pγ48.34A monoclonal antibodyconstructed by the present inventors.

FIG. 5B shows the non-specific binding of a PPAR gamma-specificmulticlonal antibody (cat. 516555 produced by Calbiochem.).

FIG. 5C shows the non-specific binding of a PPAR gamma-specific antibody(sc-7273 produced by Santacruz).

FIG. 6 schematically shows the principle of ELISA (enzyme-linkedimmunosorbent assay) for detecting PPAR regulators related toinflammation, cancer and metabolic diseases, using a monoclonal antibodyof the present invention, GST-fused PPAR gamma protein and His-fusedSRC-1 (steroid receptor coactivator).

FIG. 7A shows that the binding between SRC-1 and PPAR gamma2 isdependent on PPAR gamma concentration.

FIG. 7B shows that the binding between PPAR and SRC-1 is increased bythe addition of indomethacin. From the fact the binding between the twoproteins is increased in a ligand concentration-dependent manner at thesame protein concentration, it can be found that ELISA using a PPARgamma-specific antibody can be a good way for detecting regulatorsrelated to diseases, such as inflammation, cancer and metabolicdiseases.

FIG. 8 shows that the specific binding between SRC-1 and PPAR gamma2 isincreased by PPAR gamma2 -specific transformation. From FIG. 8, it canbe found that the use of ligands at concentrations of 1 μM, 10 μM and100 μM is most suitable, the use of His-PPAR provides better resultsthan using GST-PPAR gamma, and the binding between PPAR gamma and SRC-1is increased depending on the concentration of ligands.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will hereinafter be described in further detail byexamples. It should however be borne in mind that the present inventionis not limited to or by the examples.

Example 1 Preparation of Expression Vector Producing PPRA GammaRecombinant Protein, and isolation and Purification of RecombinantProtein

To obtain a PPAR gamma recombinant protein, a human PPAR gamma2 cDNAgene was used. This gene was obtained by isolating mRNA from humanlipoma and then amplifying it by reverse transcription-PCR. Anexpression vector pET28a (Novagen) was cleaved with BamH I and Xho Irestriction enzymes, and the gene was ligated into the vector by lagaseat 16° C. for 5 hours, thereby producing an expression vector having thePPAR gamma2 gene.

The expression vector pET28a, which can express a 6-histine-tagged PPARgamma protein, was transformed in E. coli BL21(DE3), and thenshake-cultured at 37° C. in LB medium containing 100 μg/ml kanamycinantibiotic. When the absorbance at 600 nm reached 0.4-0.6, the culturesolution was added with 1 mM IPTG (isopropylthio-β-D-galactoside), andcultured overnight at 20° C., thereby inducing the expression of thegene. The resulting culture solution was centrifuged at 4° C. and 6,000rpm for 20 minutes, and the transformed E. coli was collected. Thecollected E. coli was completely disrupted in cell lysis buffer (20 mMTris, pH 7.4, 300 mM NaCl, 10 mM imidazol, 5 μg/ml aprotinin, 100 μMPMSF) with an ultrasonic homogenizer. The disrupted solution wascentrifuged at 4° C. and 12,000 rpm for 30 minutes, and the supernatantwas collected. The collected supernatant was loaded onto NTA agarose(Peptrone) beads having an affinity for the histidine residues so thatHis-PPAR gamma2 was bound to the beads. To reduce non-specific binding,the beads were washed three times with washing solution (20 mM Tris, pH7.4, 300 mM NaCl, 20 mM imidazol). The recombinant protein bound to thebeads was separated with eluant (20 mM Tris, pH 7.4, 300 mM NaCl, 200 Mimidazol). The eluted solution was separated by electrophoresis on 12%SDS-PAGE gel to examine the eluted protein. The results are shown inFIG. 1. As shown in FIG. 1, it could be found that the protein wasHis-PPAR gamma2 recombinant protein with a 55 kDa molecular weight.

Example 2 Isolation and Purification of GST-Fused PPAR Gamma2 andHis-SRC-1 Recombinant Proteins

To produce a GST-PPAR gamma2 recombinant protein, there was used E. coli(pGEX4T-1/PPARγ 2; KCTC 10190BP) which had been transformed withexpression vector pGEX4T-1 (Pharmacia) cloned with the PPAR gamma2 geneof Example 1.

Under conditions of over-expression and efficient production of arecombinant protein, E. coli was cultured. The cultured E. coli wasdisrupted with ultrasonic waves, and centrifuged and the supernatant wascollected. A soluble fraction of the supernatant was directly used.

Meanwhile, a SRC-1 protein was obtained as follows.

E. coli (pET28a/SRC-1; KCTC 10191BP) producing a His-tagged SRC-1protein was cultured, disrupted with ultrasonic waves, and thencentrifuged, and the supernatant was collected and loaded onto NTAagarose (Peptron Inc., Daejeon, Korea) beads so that His-PPAR gamma2 wasbound to the beads. To reduce non-specific binding, the beads werewashed three times with cell lysis buffer (20 mM Tris, pH 7.4, 300 mMNaCl, 20 mM Imidazol). The recombinant protein bound to the beads wasseparated with eluant (200 Tris, pH 7.4, 300 mM NaCl, 200 mM Imidazol)and purified. The expression condition and method as described abovewere the same as Example 1, and thus, a method for examining the bindingbetween PPAR gamma2 and SRC-1 was established and could be used as anefficient screening system for the design and development of newsubstances which can influence the binding between PPAR gamma2 andSRC-1.

Example 3 Mouse Immunization

To obtain an immunized mouse required for the production of a hybridomacell line, His-PPAR gamma2 produced in Example 1 was dialyzed againstphosphate solution for 12 hours, and the protein concentration wasquantified to 25 μg/100 μl by the Bradford method. Then, it wasemulsified with an equal volume of a Freund's complete adjuvant andinjected into the abdominal cavity of a Balb/c mouse (six weeks old).After two weeks, a His-PPAR gamma2 recombinant protein of the sameamount as the first inoculation, which had been mixed with the sameamount of a Freund's incomplete adjuvant, was injected to the mouse oncea week. At 3 or 4 days after the last injection, a small amount of bloodwas collected from the mouse tail, and measured for antibody titer byenzyme-linked immunosorbent assay (ELISA). Before cell fusion, the mouseadditionally received one injection.

Example 4 Cell Fusion

The mouse immunized according to Example 3 was fractured at its cervicalvertebral. And the spleen was drawn from which fat tissues were removedand finely ground with a homogenizer. The resulting material wascentrifuged in RPMI-1640 medium, and spleen cells were collected. Atthis time, to make the spleen cells pure, leukocyte lysis buffer wasused and the spleen cells were sufficiently washed two times withRPMI-1640 medium.

Meanwhile, at two weeks before cell fusion, NS-1 myeloma cells, parentcells for cell fusion, were cultured in RPMI-1640 medium containing 10%fetal bovine serum (FBS). The NS-1 cells were washed twice in RPMI-160medium. The spleen cells and the parent cells were counted and mixed atthe ratio of 7:10. After mixing, the cells were precipitated bycentrifugation. The precipitate in a centrifuge tube was dispersed bypatting it with a finger, and lightly shaken, adding 1 ml polyethyleneglycol over one minute. Then it was filled with 50 ml of RPMI-1640medium, centrifuged and washed two times. The resulting precipitate wasre-suspended in 20-40 ml of isolation medium (HAT medium) containing 10%fetal bovine serum. 200 μl of the suspension was added to each well of a96-well plate, and then cultured in a CO₂ incubator at 37° C.

Example 5 Screening of Hybridoma Cell Line Producing Monoclonal Antibody

The cells fused in Example 4 were cultured for about two weeks, andthen, among the produced fusion cells, fusion cells secreting a specificantibody for PPAR gamma were screened.

A GST-fused PPAR gamma2 recombinant protein was isolated and purified.The purified protein-was used as an antigen in enzyme-linkedimmunosorbent assay (ELISA). The use of the GST-fused PPAR gamma2recombinant protein is to eliminate fusion cells secreting ahistidine-specific antibody, since a His-tagged PPAR gamma2 recombinantprotein was used as an antigen upon immunization of the mouse. TheGST-fused PPAR gamma2 recombinant protein was prepared in a similarmanner to the His-tagged protein, using expression vector pGEX4T-1(Amersham Pharmacia) as described in Example 2. 50 μl (8 (g/ml) of theGST-fused PPAR gamma2 recombinant protein was loaded onto each well of amircotiter plate, and unreacted antigens were blocked with skimmed milk.Then, 50 (1 of the culture solution of fusion cells was added to eachwell of the plate and allowed to react at room temperature for one hour.Then it was washed three times with phosphate buffer saline (containing0.05% Tween-20), and goat anti-mouse IgG-horseradish peroxidase (HRP:Sigma) was added. The mixture was allowed to react at room temperaturefor one hour and washed, and peroxidase substrate (OPD) was added. Thecolor reaction was measured at the absorbance at 490 nm. On the basis ofthe experimental results, fusion cells secreting an antibody having highbinding force to the PPAR gamma2 recombinant protein were screened, andthe screened cells were subjected to the above processes several times.Thus, among various fusion cells, a fusion cell population, which hasthe highest binding force and specifically reacts, was re-screened. Thescreened fusion cell was diluted to a monoclone by limiting dilution,thereby discovering a hybridoma cell line producing a monoclonalantibody originated from one cell. This hybrodoma was deposited underthe accession number KCTC 10482BP with the Korean Collection for TypeCultures (KCTC). The hybridoma cell line (KCTC 10482BP) producing amonoclonal antibody was cultured, and antibody titer was determined byenzyme-linked immunosorbent assay (ELISA). The results are shown in FIG.2A.

Furthermore, the immunoglobulin isotype of the monoclonal antibodyproduced in the hybridoma cell line was determined using an Immuno-Type™Mouse Monoclonal Antibody Isotyping Kit (BD Bioscience). As shown inFIG. 2B, it could be found that the immunoglobulin isotype of thespecific monoclonal antibody for PPAR gamma was G2a.

Example 6 Mass Production of Monoclonal Antibody

Example 6 is given to illustrate the mass production of a specificmonoclonal antibody for PPAR gamma from the hybridoma cell lineestablished in Example 5.

100 (1 of a Freund's incomplete adjuvant was injected into the abdominalcavity of an experimental mouse (Balb/c). After one week, 5×105 fusioncells were injected into the mouse, and ascitic fluid was collected whenthe abdominal cavity was swollen up. Since the ascitic fluid containedgrown fusion cells at high concentration, it was centrifuged at 10,000rpm to precipitate the fusion cells, and only the supernatant wascollected. The supernatant was separated by Protein A agarose affinitycolumn (Bio Rad), dialyzed against phosphate buffer and stored at −70(C.

Example 7 Confirmation of Antigen-Antibody Reaction of InventiveMonoclonal Antibody in Recombinant Protein, Cell Line and Tissue

In this example, using a specific monoclonal antibody for PPAR gammaobtained in Example 6, a 55 kDa PPAR gamma recombinant protein presentin a recombinant protein, a cell line and a tissue was subjected toWestern blotting. In this case, E. coli lysate was used from which humanand mouse His-tagged PPAR gamma2 recombinant proteins had not been notisolated and purified.

The E. coli lysate was centrifuged. The supernatant was suitably dilutedand separated by 12% SDS-PAGE, and the separated protein was transferredto a membrane filter. To reduce non-specific reaction that otherproteins show in the membrane filter, the membrane was blocked with 5%skimmed milk for 2 hours. Then the specific monoclonal antibody for PPARgamma developed by the present inventors was added to the membranefilter and allowed to react for one hour. Then it was washed withphosphate buffer saline (containing 0.05% Tween-20), and goat anti-mouseIgG-alkaline phosphatase (AP) (Sigma) was added and allowed to react atroom temperature for one hour. Then, the membrane filter wassufficiently washed and the color was developed with alkalinephosphatase substrate (Promega). FIG. 3A shows that the monoclonalantibody specifically reacts with human PPAR gamma without binding toHis, and FIG. 3B shows that the monoclonal antibody of the presentinvention does not react with skimmed milk and also bovine serum albumin(BSA). Some of other commercially available PPAR gamma antibodies reactwith BSA. As a result, it could be found that the monoclonal antibody ofthe present invention does not have non-specific reaction and was veryspecific for PPAR gamma.

Meanwhile, it could be found that PPAR gamma was expressed in a mouse3T3-L1 cell line, a fat cell line. To confirm this expression, Westernblotting was used. As the 3T3-L1 cell line, cells which had beendifferentiated with 1 μg/ml insulin and 30 uM indomethacine and had notbeen differentiated were used. The use of these cells is to confirm thatPPAR gamma is expressed in the differentiated cell line at a largeramount. An eluate containing the cytoplasm and nucleus of the cells wasobtained, and an equal amount of the protein was separated by 12%SDS-PAGE, and subjected to Western blotting.

Color development was performed by ECL regent (Pharmacia-Amersham). FIG.4A shows that PPAR gamma is expressed in the differentiated mouse.

FIG. 4B shows that antigen-antibody reaction of the specific monoclonalantibody for PPAR gamma occurs in the mouse 3T3-L1 cell line byimmunoprecipitation.

The experiment in FIG. 4B was carried out as follows. 5 μl of thespecific monoclonal antibody for PPAR gamma was added to the eluatecontaining the cytoplasm and intranuclear protein of the differentiatedand non-differentiated cells, and allowed to react at 4° C. for onehour. And, the mixture was added to 10 μl protein-A agarose beads andallowed to further react at 4° C. for 30 minutes. An antigen-antibodycomplex bound to the beads was washed three times and purified bycentrifugation. Then, the complex was added to 5-fold sample-loadedbuffer and warmed in boiling water for 5 minutes. And, it was separatedby 12% SDS-PAGE, transferred to a membrane filter and subjected toWestern blotting with the specific monoclonal antibody for PPAR gamma.

As described above, it can be found that the PPAR gamma-specificmonoclonal antibody produced by the present inventors can be used inWestern blotting and also immunoprecipitation.

To compare the specificity and binding of the PPAR gamma specificmonoclonal antibody (Pγ48.34A) of the present invention to those ofcommercially available PPAR gamma-specific antibodies, the followingtest was conducted.

First, various tissues of a mouse were collected and lysated in celllysis buffer (20 mM Hepes, 300 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1% NP40,pH 7.5). The lysated cells were centrifuged at 12,000 rpm, and thesupernatant was collected. The protein concentration of the cell lysatewas determined by Bradford protein assay, and each 100 μg of the celllysate was loaded for 12% SDS-PAGE. After electrophoresis, it wassubjected to Western blotting with the PPAR gamma-specific Pγ48.34Aantibody of the present invention, a specific multiclonal antibody forPPAR gamma (cat. 516555 made by Calbiochem company), and a specificmonoclonal antibody for PPAR gamma (sc-7273 made by Santacruz company).The test results are shown in FIG. 5. From FIG. 5, it could be foundthat the Pγ48.34A monoclonal antibody of the present invention showedsignificantly reduced non-specific binding and more excellent binding ascompared to other company's products.

Example 8 Mechanism of PPAR Gamma2 and SRC-1 Proteins in Cells andPrinciple of ELISA Using It

PPAR gamma2 is specifically expressed in fat cells, binds to RXR alphato form heterodimers, and binds to PPRE (PPAR response elements. Thiscomplex is a transcriptional regulator which binds to transcriptionalfactors in a complex manner to promote the transcription of a targetgene. Its transcriptional activity is determined depending on whether ithas hydrophobic low molecules (agonists) or not.

When 15d-PGJ2 or TZDs, currently known PPAR gamma ligands, binds to PPARgamma, their binding force to SRC-1 protein, a coactivator, is reduceddue to the transformation of PPAR gamma protein itself. These ligandsincrease the half-life of PPAR gamma in cells, and further increase thetranscription of a target gene by binding to coactivators. The ligandscapable of increasing the binding force between PPAR gamma and SRC-1 arecalled agonists whose possibility for use as anti-cancer agents andanti-inflammatory agents is being increased.

Particularly, TZDs are marketed as agents for treating type 2 diabetes.

By applying such intracellular mechanisms in ELISA, the presentinventors have developed a method for detecting PPAR gamma agonists orPPAR gamma ligands, which is more easily conducted and allows manysamples to be received. As shown in FIG. 6, this method comprisescoating SRC-1 proteins on an ELISA plate, treating E. coli lysatecontaining GST-PPAR gamma with a sample, and determining the bindingbetween SRC-1 and PPAR gamma using Pγ48.34A, a specific monoclonalantibody for PPAR gamma.

If substances which can be PPAR gamma agonists are present in thetreated samples, the binding shown by an ELISA reader will be a highernumerical value than a control group. On the other hand, if there areantagonists inhibiting the binding therebetween, he binding shown by theELISA reader will be a lower numerical value than the control group.

Example 9 Effect of PPAR Gamma2 and Ligand Concentrations on Bindingbetween SRC-1 and PPAR Gamma2 Proteins in ELISA

To examine the effect of PPAR gamma2 protein concentration on thebinding between SRC-1 and PPAR gamma2 proteins, the following test wasconducted. As shown in FIG. 7A, 0.8 μg/ml SRC-1 was coated on a plate,and E. coli lysate containing GST-PPAR gamma2 was diluted 512-, 256-,128-, 64-, 32-, 16-, 8- and 4-fold and added to the plate. Also, thisprocedure was repeated three times, and a GST-fused protein which hadnot been bound to SRC-1 protein was used as a control group. As shown inFIG. 7A, it could be found that the binding between SRC-1 and PPARgamma2 proteins in ELISA was dependent on the concentration of PPARgamma2. Furthermore, to examine the effect of PPAR gamma2 ligandconcentration on the binding between SRC-1 and PPAR gamma2 proteins, thefollowing test was conducted. 8 μg/ml SRC-1 was coated on a plate, andE. coli lysate was diluted 64-fold and added to the plate. Then,indomethacin, a PPAR gamma2 ligand, was added to the plate at adifferent concentration.

The results are given in FIG. 7B. As shown in FIG. 7B, it could be foundthat the binding was dependent on the concentration of indomethacin, aPPAR gamma2 ligand. The results of FIG. 7B were given as an average ofthe results of more three tests.

Example 10 Establishment of System Capable of Detecting PPAR Ligandsusing in vitro Binding between Soluble Fractions of GST-PPAR Gamma2 andHis-SRC-1 Proteins

The binding between SRC-1 protein and PPAR gamma in a soluble fractionwas measured by ELISA.

His-SRC-1 protein isolated from E. coli and eluted from nickel beads,and purified PPAR gamma-fused protein produced in E. coli, were used asantigens.

His-SRC-1 protein was diluted with 0.1 M sodium carbonate buffer (pH9.6) to a concentration of about 800 ng/well. 100 μl of the proteindilution was loaded into each well of an ELISA plate, and coated on theplate overnight at 4° C. The coated plate was washed three times withPBS (containing 0.05% Tween-20), and added with 3% skimmed-PBST solutionand left to stand overnight at 4° C. or for 2 hours at room temperature.A soluble fraction of GST-fused PPAR gamma2 was diluted 32-fold, and 50μl of the dilution was added to each well of the plate. Indomethacin, aPPAR gamma2 agonist, was added at 1 μM, 10 μM and 100 μM.

The plate was subjected to binding reaction at room temperature for onehour with slow stirring, and washed five times with PBS (containing0.05% Tween-20). To determine the binding between the His-SRC-1 proteinand the GST-fused PPAR gamma2 protein, a monoclonal antibody (P48.34A)which had been developed by the present inventors was 2,000-fold dilutedwith PBST and used as a primary antibody. An anti-mouse IgG-HRP antibodywhich had been 2,000-fold diluted was used as a secondary antibody. As acontrol group, DMSO (dimethylpolysiloxane) in which PPAR gamma2 agonistshad been dissolved was added as a negative control for PPAR gamma2ligands. The control group showed no significant change in theabsorbance at 490 nm. The test results are shown in FIG. 8. From FIG. 8,it could be found that the specific binding between SRC-1 protein andthe PPAR gamma2 protein was promoted by ligand-specific conformation.Using this principle, an ELISA system capable of screening PPAR gamma2-specific ligands was established.

Example 11 Utility of ELISA Detection Method

The detection method according to the present invention differs from theprior detection method. The present invention provides a screeningsystem which does not use radioactive isotopes, allows an increasednumber of samples to be used at the same time, and shows results in amore rapid manner. In the present invention, an antibody is used insteadof radioactive isotopes, a 96-well plate is used to allow many kinds ofsamples to be used at the same time, and color development using anenzyme is used so that results can be shown in a more rapid manner thanthe prior method.

By virtue of such convenience and rapidness, the detection method of theprevent invention will be highly effective in detecting ligands invarious functional foods and plant extracts. The detection method of thepresent invention is a method for detecting PPAR gamma ligands closelyrelated to cancer inhibition, inflammation inhibition, obesity,diabetes, lipid metabolism and cancer. Thus, according to this method,many substances which show effects against such diseases can beprimarily detected in a rapid and easy manner.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides a method forprimarily detecting regulators related to cancer, inflammation, obesityand diabetes, which is based on the binding between transcriptionalfactor SRC-1 and PPAR gamma, a nuclear receptor known as having aneffect on anti-inflammation and anti-cancer. This method is easilyconducted, allows many samples to be applied at the same time, and doesnot utilize radioactive isotopes. Thus, this method will beadvantageously applied for the development of agents for the improvementand treatment of cancer, inflammation, obesity and metabolic diseases.

1. A Pγ48.34A cell line (KCTC 10482BP), which produces a monoclonalantibody whose immunoglobulin isotype is G_(2a), the monoclonal antibodyhaving specific immunoreactivity for human and mouse PPAR gammaproteins.
 2. The Pγ48.34A cell line of claim 1, wherein the PPAR gammaproteins are PPAR gamma1 or PPAR gamma2.
 3. A monoclonal antibodyproduced by the cell line of claim
 1. 4. A method for detecting aregulator related to cancer, inflammation and metabolic diseases, themethod comprising the steps of: (a) coating a PPAR gamma coactivator ona plate; (b) adding a sample containing a PPAR gamma protein and aregulator related to cancer, inflammation and metabolic diseases to theplate; (c) adding the monoclonal antibody of claim 3 to the plate sothat the antibody binds to the PPAR gamma protein; (d) adding anantibody having immunoreactivity for the monoclonal antibody and labeledwith an enzyme to the plate; and (e) detecting the label and measuringthe concentration of the PPAR gamma protein, thereby determining if theregulator related to inflammation, cancer and metabolic diseases ispresent in the sample.
 5. The method of claim 4, wherein the PPAR gammacoactivator is SRC-1.
 6. The method of claim 4, wherein the sample isplant extract, functional food or a pharmaceutical composition.
 7. A kitfor detecting a regulator related to cancer, inflammation and metabolicdiseases by immunoassay, the kit comprising: a coactivator proteincoated on a plate; a PPAR gamma protein capable of binding to thecoactivator protein; a monoclonal antibody having specificimmunoreactivity for the PPAR gamma protein; and an antibody labeledwith an enzyme and having immunoreactivity for the monoclonal antibody.8. The kit of claim 1, wherein the coactivator protein is SRC-1.